Chronic Hepatitis C virus (HCV) infection causes hepatitis and hepatocellular carcinoma (HCC). The mechanisms of HCV-induced carcinogenesis are likely to include direct oncogenic stimuli by viral proteins and indirect tumorigenic effects from inflammation. Viral genes that may induce direct oncogenic stimuli include core, NS3, and NS5A, and inflammation could be modulated by NS3 and NS5A. HCV's genetic variation could clearly affect its oncogenic potential, but there is no consensus on how HCV's high diversity may affect oncogenesis because viral genetic analyses have been limited to detailed inspection of small regions of the genome or to characterizing HCV's diversity at the insensitive level of the genotype. Furthermore, biochemical analyses attributing oncogenic potential to HCV proteins have yielded conflicting results, possibly because few have addressed HCV's genetic diversity. Therefore, we will perform a comprehensive analysis of the effect of HCV's genetic variation on carcinogenic potential employing coordinated genetic and biochemical approaches. Hypothesis: HCV genetic variation contributes to development of HCC by modulating function of viral proteins that promote tumor growth and/or moderate inflammation. Aim 1. Correlate HCV sequence variation with development of HCC. We will sequence the complete HCV protein coding region from 20 patients who developed HCC and from 20 random population controls without HCC and the presence of sequence patterns that correlate with HCC will be evaluated. Aim 2. Determine how genetic variation affects putative tumor promoting activities of HCV proteins. We will express variant core, NS3/4A, and NS5A genes from HCC and control subjects in Aim 1 and measure their ability to transform cells and to alter transcription of cell-cycle regulatory genes. We will also assess other HCV genes for which genetic correlations with HCC are observed in Aim 1. Aim 3. Determine how genetic variation affects the ability of HCV proteins to moderate inflammation. We will express variant NS3/4A and NS5A genes from HCC and control subjects in Aim 1 and measure their activities proposed to counteract inflammatory responses. We will also assess other HCV genes for which novel genetic correlations with HCC are observed in Aim 1. These data will clarify the mechanism(s) by which HCV promotes cancer, and hence may identify approaches to delay or halt development of HCC. They may also lead to greater ability to identify patients at highest risk for development of HCC through discovery of viral motifs associated with HCC.